Recent advances in biotechnology have made it possible to produce a variety of peptides for pharmaceutical applications using recombinant DNA and synthetic techniques. AC-100, also known as Dentonin®, is a therapeutically active peptide shown to stimulate proliferation, differentiation, and mineralization of human osteoblasts (Nagel et al. (2004) J. Cell. Biochem. 93(6):1107-14; U.S. Pat. No. 6,911,425; U.S. Pat. No. 7,078,021 and U.S. Pat. No. 7,160,862). AC-100 has shown bone formation activities in vivo (Hayashibara et al. (2004) J. Bone and Mineral Res. 19(3):455-62; Lazarov et al. ((2004) ASBMR Abs.); and has also demonstrated stimulation of the proliferation of human dental pulp cells in vitro (Liu et al. (2004) J. of Dental Res. 83(6):496-99); as well as formation of new dentin in human dental defects in a clinical trial (Lazarov et al. (2006) IADR Abs.).
Accordingly, AC-100 is useful in treating or preventing conditions associated with skeletal loss or weakness, increasing the number and biological activity of osteoblasts, odontoblasts, and other hard tissue forming cells that assist in forming skeletal and dental tissues and promoting regeneration of bones, teeth, and/or cartilage. The described therapeutic peptide may be administered, inter alia, in the treatment of bone defects and breakage, cartilage regeneration, and the stimulation of dental pulp cells to promote hard tissue formation.
Because therapeutic peptides may be larger and more complex than traditional organic and inorganic drugs (i.e., possessing multiple functional groups in addition to possibly complex three-dimensional structures), the formulation of such peptides poses special problems. For a peptide to remain biologically active, a formulation must preserve intact the conformational integrity of at least a core sequence of the peptide's primary structure while at the same time protecting the peptide's multiple functional groups from degradation. Degradation pathways for peptides can involve chemical instability (i.e., any process which involves modification of the peptide by bond formation or cleavage resulting in a new chemical entity) or physical instability (i.e., changes in the higher order structure of the peptide). Chemical instability can result from deamidation, racemization, hydrolysis, oxidation, beta elimination or disulfide exchange. Physical instability can result from denaturation, aggregation, precipitation or adsorption, for example. The three most common peptide degradation pathways are peptide aggregation, deamidation and oxidation. Cleland et al. (1993) Critical Rev. in Therapeutic Drug Carrier Sys. 10(4):307-377.
Freeze-drying is a commonly employed technique for preserving peptides which serves to remove water from the peptide preparation of interest. Freeze-drying, or lyophilization, is a process by which the material to be dried is first frozen and then the ice or frozen solvent is removed by sublimation in a vacuum environment. An excipient may be included in pre-lyophilized formulations to enhance stability during the freeze-drying process and/or to improve stability of the lyophilized product upon storage. Pikal, M. (1990) Biopharm. 3(9):26-30 and Arakawa et al. (1991) Pharm. Res. 8(3):285-291.
It is an object of the present invention to provide a lyophilized peptide formulation, which is stable upon storage and delivery at relatively high temperatures, and which may later be reconstituted to generate an aqueous drug product. It is a further object to provide a stable reconstituted peptide formulation, which is suitable for injectable, such as by intravenous and/or subcutaneous administration, for example. Accordingly, in certain embodiments, it is an object to provide a formulation that is stable as a lyophilized product when stored at elevated temperatures for several months at a time.
Another object of the present invention is to provide a lyophilized peptide formulation, which can be sterilized by radiation. If the peptide in the formulation is susceptible to degradation or structural modification by radiation, the peptide formulation must be sterilized by filtering through sterile filtration membranes prior to, or following, lyophilization and reconstitution. A formulation that enables the terminal sterilization by radiation, e.g., gamma irradiation, would significantly simplify the final sterilization process of the formulated drug.